Dendritic and hyperbranched polymers for cellular encapsulation and functionalization

ABSTRACT

A protective coating for covering a biological material, the protective coating having a plurality of interconnected layers covalently bonded to each other. The plurality of interconnected layers can include at least one hyperbranched polymeric material, and at least one dendrimer. A method of forming a protective coating for covering a biological material, the method can include depositing a plurality of interconnected layers, which are covalently bonded to each other. The plurality of interconnected layers can include at least one hyperbranched polymeric material, and at least one dendrimer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 12/633,270, titled “Cross-linked Alginate-Polyalkylene GlycolPolymer Coatings for Encapsulation and Methods of Making the Same” filedon Dec. 8, 2009, which claims priority from U.S. Provisional PatentApplication No. 61/120,711, titled “Crosslinked Alginate-PEG Polymersfor Cellular Encapsulation” and filed on Dec. 8, 2008. Both U.S. patentapplication Ser. No. 12/633,270 and U.S. Provisional Patent ApplicationNo. 61/120,711 are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is generally directed toward crosslinkedalginate-polyalkylene glycol polymer coatings for encapsulation ofparticles and cells and methods of making the same. More specifically,the present invention relates to polymeric functional materials forconformal coating and functionalization of biological surfaces.

BACKGROUND OF THE INVENTION

Cellular encapsulation has been a subject of research for severaldecades as a means to mask transplanted cells from the in vivo host. Bycoating the cells with a highly biocompatible layer, surface antigens,inflammatory proteins, and other agents that may instigate animmune/inflammatory response may be dampened or eliminated. In order todevelop a reasonable method to encapsulate a cell, the process must notbe detrimental to the cells and result in a highly biostable andbiocompatible coating. Cellular encapsulation through the use of highlypurified alginate has shown significant promise. In fact, clinicaltrials using islets within alginate capsules in the absence of animmunosuppression regimen are ongoing worldwide. However, to date,studies using this approach have failed because 1) the resulting gelsare unstable; and 2) the resulting coating prevents adequate nutrientdelivery for highly metabolically active cells, such as islets ofLangerhans.

Various permutations of alginate and/or PEG encapsulation of mammaliancells has been patented, including U.S. Pat. Nos. 4,353,888; 4,673,566;4,689,293; 4,806,355; 4,923,645; 5,762,959 and 5,766,907). However, mostof these methods produce alginate gels that degrade over time due toleakage of divalent cations.

Additional research has been conducted to attempt to increase thestability of alginate and/or PEG-based gels through the use of covalentcross-linking. For example, free radical polymerization generation ofcapsules to entrap mammalian cells has been disclosed in U.S. Pat. Nos.5,334,640; 5,410,016; 5,700,848; 5,705,270; and 6,258,870. However,these techniques have not proven useful because these methods inducemoderate to severe cell damage, particularly for cells that arevulnerable to oxidative stress, such as islets of Langerhans.

Layer-by-layer coating of thin films has multiple biomedicalapplications, from enhancing biocompatibility of an inert implant, toimmune-camouflage of a cellular transplant, to local drug delivery.

Layer-by-layer encapsulation has been used for temporal drug deliveryfor multiple applications. Drug microcrystals, proteins, or enzymes canbe encapsulated and the rate of release can be modified through theproperties of the coating and their subsequent thickness. Furthermore,the overall polymer load is substantially reduced, given that thethickness of the coating can be intricately controlled. Agents may beencapsulated in their crystalline form, or via infiltration into amatrix that is subsequently coated. Of note, recent publishedapplications of layer-by-layer methods for drug delivery include:ibuprofen, furosemide, doxorubicin, rifampicin, curcumin, or evenpeptides or enzymes.

SUMMARY OF THE INVENTION

Polymers according to various embodiments have flexibility in theirhydrophobicity (through the phosphine/MCT group), their charge (positiveor neutral), and their overall thickness through layer assembly. Thisprovides a versatile platform for application to numerous drugs, such asthose mentioned in the examples given above. Furthermore, the ease inwhich the surface of these coatings may be functionalized with variousantibodies or antigens permits for the targeting and, if desired,phagocytosis, of these drug eluting materials to specific cells.

In one embodiment, the invention is drawn to a biocompatible capsulehaving a biological material and a covalently stabilized coatingencapsulating the biological material. The covalently stabilized coatingcan be formed by reacting (i) alginate—[polyalkylene glycol(PAG)-X¹]_(n), and (ii) multi-functional PAG-X², to form covalent bonds,wherein n is an integer greater than 0, a first one of X¹ and X² is N₃,and a second one of X¹ and X² is selected from the group consisting of:

(b) —C≡C; and

wherein R¹═CH₃, CH₂CH₃, CH(CH₃)₂, or

R²═N(CH₃)₂, OCH₃, OH, CH₃, H, F, Cl, Br or NO₂, and R³═OCH₃, CH₃, H, F,C₁ or NO₂.

The biological material can include a material selected from the groupconsisting of cells, pharmaceuticals, biological agents, biopolymers,RNA, DNA and fragments of DNA or RNA. The cells can be islets ofLangerhans.

The covalently stabilized coating can include a plurality of monolayers.The covalently stabilized coating can be covalently bonded to thebiological material. The plurality of monolayers alternate betweenmonolayers of Alginate—[PAG-X¹]_(n) reaction products and monolayers ofmulti-functional PAG-X² reaction products. The multi-functional PAG-X²comprises a multi-arm PAG-X² having at least three PAG-X² arms.

The alginate—[polyethylene glycol (PAG)-X¹], molecule, themulti-functional PAG-X² molecule, or both, can also include anadditional terminal ligand, X³. The additional terminal ligand, X³, canbe selected from the group consisting of proteins, imaging labels,nanoparticles, biopolymers, RNA, DNA, and fragments of RNA or DNA.

The coating can be covalently bonded to the biological material byreacting a first terminal ligand of compound (A) with amino groups on asurface of the biological material. Compound (A) can include:

the first terminal ligand comprising

anda second terminal ligand comprising a ligand selected from the groupconsisting of:

(b) —C≡C;(c) —N₃; and

In compound (A), R¹ can be CH₃, CH₂CH₃, CH(CH₃)₂, or

R² can be N(CH₃)₂, OCH₃, OH, CH₃, H, F, Cl, Br or NO₂, and R³ can beOCH₃, CH₃, H, F, C₁ or NO₂.

The invention is also drawn to a method of forming the biocompatiblecapsule described above by reacting (i) alginate—[polyalkylene glycol(PAG)-X¹]_(n), (ii) multi-functional PAG-X², or (iii) both, in anaqueous solution containing the biological material. The reacting stepcan proceed without a free-radical initiator. The reacting step canproduce a covalent bond resulting from reacting an X¹ ligand of saidalginate—[PAG-X¹]_(n) with an X² ligand of said multi-functional PAG-X².The reacting step can include adding molecular monolayers of eitheralginate—[PAG-X¹]_(n) or multi-functional PAG-X².

According to various other embodiments, the coatings can have a highlybranched architectural design. Such coatings can be used to buildprotective coatings on cells, cell clusters, or other bioactive agents,in this way allowing enhanced control over the physical, chemical, andbiological properties of the coating. In addition, the unique branchedsystem provides for a high degree of surface presentation of agents,thereby providing a novel platform for the chemoselective presentationof a large amount of agents on the surface of the coating (which couldbe highly beneficial for targeting, labeling, or as a bioactiveinterface).

One embodiment relates to a protective coating for covering a biologicalmaterial, the protective coating having a plurality of interconnectedlayers covalently bonded to each other. The plurality of interconnectedlayers can include at least one hyperbranched polymeric material, and atleast one dendrimer. The protective coating can also include at leastone biologically-active agent chemoselectively presented on a surface ofthe coating. The biologically-active agent can be selected from atargeting agent, a labeling agent, a bioactive interface, andcombinations thereof. The protective coating can have a thickness ofless than 10 nm. The biological material can be selected from aplurality of cells, a plurality of cell clusters, a plurality ofbioactive agents, and combinations thereof.

Another embodiment relates to a method of forming a protective coatingfor covering a biological material, the method can include depositing aplurality of interconnected layers, which are covalently bonded to eachother. The plurality of interconnected layers can include at least onehyperbranched polymeric material, and at least one dendrimer. The methodcan also include applying at least one biologically-active agent onto asurface of the coating. The biologically-active agent can be selectedfrom a targeting agent, a labeling agent, a bioactive interface, andcombinations thereof. The protective coating can have a thickness from 1nm to up to 1000 nm. The biological material can be selected from asingle cell, a plurality of cells, a plurality of cell clusters, aplurality of bioactive agents, and combinations thereof.

The coating systems according to various embodiments can be particularlyapplicable to the coating of drugs for stabilization or targeting—giventhe benign nature of the coating and the resulting coating stability.

These and other embodiments are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A fuller understanding of the present invention and the features andbenefits thereof will be obtained upon review of the following detaileddescription together with the accompanying drawings, in which:

FIG. 1 is a schematic showing a gel and lock mechanism useful forproducing encapsulated particles of the invention.

FIGS. 2(A)-(C) show confocal images of encapsulated Lewis rat isletsstained using a Live/Dead fluorescent kit where live cells are labeledgreen and dead cells are labeled red.

FIG. 3(A)-(C) show confocal images of encapsulated human islets stainedusing a Live/Dead fluorescent kit where live cells are labeled green anddead cells are labeled red.

FIG. 4 shows confocal image of alginate-azide microcapsules withcovalent bond linkage to DiP-PEG-carboxyl fluorescein linker,illustrating the capacity of the gels to covalently bond with imageenhancement agents.

FIGS. 5(A)-(C) show beads prepared with 1.5 wt % of molecule [1] andmolecule [3] via (A) ionic bonding (with Ba²⁺ and no molecule [3]), (B)both ionic and covalent bonding, and (C) covalent bonding (after removalof Ba²⁺ with EDTA).

FIGS. 6(A)-(C) show fluorescent microscope images of glass microcarrierbeads functionalized with 4-aminobutyl-triethoxy-silane and reacted withfunctionalized polymers.

FIGS. 7(A)-(F) show confocal fluorescent and light transmission imagesof nano-layering on glass microcarrier beads (170 to 210 μm glass beadswere functionalized with 4-aminobutyl-triethoxy-silane) reacted withfunctionalized polymers.

FIGS. 8(A)-(D) show Atomic Force Microscopy (AFM) images, where FIGS.8(A) and (B) are height and deflection images, respectively, of Corningamino glass functionalized with a layer of Azide-PEG-NHS [5]; and FIGS.8(C) and (D) are height and deflection images, respectively, of Corningamino glass functionalized with a layer of Azide-PEG-NHS [5] followed bya layer of 4DiP-PEG [3].

FIG. 9 shows a schematic representation of covalently linked layers oftriarylphosphine PEG active ester (base layer), azido-alginate(interconnecting layer) and bi-triarylphosphine PEG (interconnectinglayer and terminal layer).

FIG. 10 shows a chemical scheme outlining the reactions for the covalentlinking of two layers (triarylphosphine PEG active ester andazido-alginate) on the islet surface.

FIGS. 11(A)-(D) show fluorescent and light transmission confocal imagesof Cytodex-3 beads treated with functionalized polymers disclosedherein.

FIGS. 12(A) and (B) show confocal images of human islets treated withfunctionalized polymers as disclosed herein.

FIG. 13 shows various three-dimensional gels, capsules, cylinders, anddisks, created using bulk cross-linking methods disclosed herein.

FIG. 14A is a diagram of a functionalized PAMAM dendrimer.

FIG. 14B is a diagram of an azido-functionalized hyperbranched alginate.

FIG. 15 is a confocal cross-section image revealing the fluorescentcapsule of 6 layers deposited around on rat pancreatic islets.

FIG. 16 is a chart showing an amount of insulin released by islets uponincubation in low (60 mg/dL), high (300 mg/dL), and low glucosecontaining buffer for 1 h each. Group 1 are the control islets (nocoating), while group 2 is the nano-scale encapsulated islets.

FIG. 17 is an electron microscopic image of a 12 layer film deposited onSi wafer.

FIG. 18 is a chart showing the film thickness increase upon depositionof a bilayer on the azido-functionalized Si wafer.

DETAILED DESCRIPTION

The invention is drawn to customized cross-linked alginate-polyalkyleneglycol (PAG) polymer coatings for encapsulating biological materials andmethods of applying the same. The polymer coatings are produced using astep-wise, chemoselective ligation scheme that does not require afree-radical initiator or elevated temperature. The techniques disclosedherein enable application of molecular monolayers of the alginate-PAGcoatings on the biological materials. These monolayer coatings can be onthe order of nanometers in thickness. It has been unexpectedlydiscovered that the coatings disclosed herein enable application of acoating that prevents rejection of coated cells by a host organism, suchas a human, without causing premature death of encapsulated cells.

In one embodiment, the invention is drawn to a biocompatible capsulethat includes a biological material and a covalently stabilized coatingencapsulating the biological material. The covalently stabilized coatingcan be formed by reacting:

alginate—[polyalkylene glycol-X¹]_(n), and

multi-functional polyalkylene glycol-X², to form covalent bonds.

In the above formulas, the molecular weight of the polyalkylene glycol(PAG) can be between 500 and 250,000 Daltons; the polyalkylene glycolcan be polyethylene glycol (PEG), polypropylene glycol (PPG) or acopolymer (PEG-PPG) thereof; n can be an integer greater than 0; a firstone of X¹ and X² can be N₃; and a second one of X¹ and X² can beselected from the group consisting of:

—C≡C; and

In ligands (a), (b) and (c) above, R¹═CH₃, CH₂CH₃, CH(CH₃)₂, or

R²═N(CH₃)₂, OCH₃, OH, CH₃, H, F, Cl, Br or NO₂, andR³═OCH₃, CH₃, H, F, C₁ or NO₂.

In some embodiments, ligands (a), (b) and (c) above, R¹═CH₃, CH₂CH₃ orCH(CH₃)₂, and R²═N(CH₃)₂, OCH₃, OH, CH₃ or H. In other embodiments, forligands (a), (b) and (c) above, R¹═CH₃, and R²═N(CH₃)₂, OCH₃, OH, CH₃ orH.

In some embodiments, n can be greater than 1, or n can be greater than3, or n can be greater than 10, or n can be greater than 20. In someembodiments, the molecular weight of the polyalkylene glycol can be 750to 100,000 Daltons, or 750 to 50,000 Daltons, or 1,000 to 25,000Daltons, or 1,000 to 15,000 Daltons. Exemplary polyalkylene glycolsinclude, but are not limited to polyethylene glycol, polypropyleneglycol, polybutylene glycol and copolymers thereof.

As used herein, “encapsulating” is used to refer to a coating thatcompletely surrounds and physically separates the encapsulated materialfrom the surrounding environment. An encapsulating coating can becontinuous and can have sufficient permeability to enable anencapsulated cell to transfer nutrients, waste, and oxygen with thesurround environment while preventing an adverse immune response by ananimal in which the encapsulated cell is implanted.

As used herein, “multi-functional” is used to refer to a molecule havingmore than one terminal ligand capable of forming a covalent bond orserving as an additional agent, for example an engineered protein, afluorescent marker, a nuclear label, or a nanoparticle. In particular,the multi-functional molecules disclosed herein generally include atleast one terminal ligand capable of forming a covalent bond viaStaudinger ligation chemistry, copper-catalyzed Click chemistry orcopper-free Click chemistry.

Although described as (i) alginate—[polyalkylene glycol-X¹]_(n) and (ii)multi-functional polyalkylene glycol-X², it should be noted that thelinkages between these molecular constituents can be direct or can bevia a linking group. For example, the polyalkylene glycol in formulas(I) or (ii) can be connected to the terminal ligand, X¹ and X²,respectively, via a linking group. Exemplary linking groups include, butare not limited to alkanes, alkenes, alkynes, ethers, esters, amines,thiols, or combinations thereof.

The alginate—[PAG)-X¹]_(n) molecule or the multi-functional PAG-X²molecule can also include an additional terminal ligand, X³. Theterminal ligand, X³, can be selected from the group including, but notlimited to, proteins, imaging labels, nanoparticles, biopolymers, RNAand DNA.

By definition, the multi-functional PAG-X² compound has at least twoPAG-X² arms. The multi-functional PAG-X² can be a multi-arm PAG-X²having at least three PAG-X² arms, at least four PAG-X² arms or at leasteight PAG-X² arms. In some embodiments, the multi-functional PAG-X² canhave two or three PAG-X² arms and one or two arms with differentfunctionalities, X³, such as a flourescein, a nanoparticle, or aPAG-fluorescein.

The biological material can be a cell, a cluster of cells,pharmaceuticals, biological agents, or a combination thereof. Generally,the biological materials will be cells or particles having a diameterless than 10 μm, or less than 1 μm, or less than 0.5 μm, or less than 50nanometers.

The biological material can be a cell or a cluster of cells. Exemplarycells that can be used in the microcapsules and methods disclosed hereininclude, but are not limited to, islets of Langerhans, dopaminesecreting cells, nerve growth factor secreting cells, hepatocytes,adrenaline/angiotensin secreting cells, parathyroid cells andnorepinephrine or metencephalin secreting cells.

The covalently stabilized coating can include a plurality of monolayersof polymer material. As used herein, “monolayers” refer to molecularlayers of polymer material (e.g., Alginate—[PAG-X¹]_(n) reactionproducts and multi-functional PAG-X² reaction products) that arecontinuous. Monolayers are generally produced by step-growth techniqueswhere one molecular layer of reactant is applied to a surface at a time,such as where one of two reactant species of a Staudinger ligation aredissolved in a solution and the other reactant species is bound to thesurface being coated. Examples of molecular monolayers are shown inFIGS. 9 and 10.

Monolayers can be contrasted with layers formed by bulk polymerization,such as free-radical polymerization or where both reactants of aStaudinger ligation are dissolved in a single solution. In bulkpolymerization, the reactions can happen in a random order and will notform molecular layers of polymer that are continuous. Rather, a layerproduced by bulk polymerization will be thicker than a molecularmonolayer and will include a mixture of the reactants without the orderof a molecular monolayer.

In one embodiment, the plurality of monolayers can alternate betweenmonolayers of Alginate—[PAG-X¹]_(n) reaction products and monolayers ofmulti-functional PAG-X² reaction products. As used herein, “reactionproducts” refer to polymers resulting when X¹ and X² react to form acovalent bond. Examples are shown schematically in FIG. 10.

In another embodiment, a bulk layer and one or more monolayers can beapplied to a biological material. For example, a monolayer can beapplied to covalently bond the coating to the cell and then a bulk layercan be applied over the base monolayer.

The covalently stabilized coating can be at least one molecule thick,e.g., at least 0.1 nanometer thick. The covalently stabilized coatingcan be less than 300 microns thick, or less than 200 microns thick, orless than 100 microns thick, or less than 50 microns thick, or less than25 microns thick, or less than 10 microns thick, or less than 1 micronthick.

The covalently stabilized coating can be covalently bonded to thebiological material. The coating can be covalently bonded to thebiological material by reacting a first terminal ligand of compound (A)with amino groups on a surface of the biological material. Compound (A)can include:

a first terminal ligand comprising

anda second terminal ligand comprising a ligand selected from the groupconsisting of:

—C≡C;—N₃; and

In compound (A), R¹ can be CH₃, CH₂CH₃, CH(CH₃)₂, or

R² can be N(CH₃)₂, OCH₃, OH, CH₃, H, F, Cl, Br or NO₂, and R³ can beOCH₃, CH₃, H, F, C₁ or NO₂.

Compound (A) can include a variety of ligands intermediate to the firstand second terminal ligand. Exemplary intermediate ligands include PAGs,such as polyethylene glycol, polypropylene glycol and copolymersthereof.

In another embodiment, the invention is drawn to a method of forming thebiocompatible capsules disclosed herein. The method can include forminga covalently stabilized coating encapsulating a biological material. Theforming step can include providing an aqueous solution having thebiological material suspended therein, where the aqueous solution alsoincludes a reactant dissolved therein. The dissolved reactant caninclude (i) alginate—[PAG-X¹]_(n), (ii) multi-functional PAG-X², or(iii) both. The method can also include reacting alginate—[PAG-X¹]_(n)with multi-functional PAG-X² in the aqueous solution, wherein n is aninteger greater than 0, a first one of X¹ and X² is N₃, and a second oneof X¹ and X² is selected from the group consisting of:

—C≡C; and

In the above formulas of (a), (b) and (c), R¹, R² and R³ have the samemeanings used throughout this disclosure. The reacting step can occurbetween approximately 40° C. and 20° C. For example, reacting step canoccur at between about 33° C. and 40° C., or at room temperature, suchas approximately 21 to 27° C.

The reacting step can produce a covalent bond resulting from reacting anX¹ ligand of the alginate—[PAG-X¹]_(n) with an X² ligand of themulti-functional PAG-X². In one embodiment, the aqueous solution caninclude either alginate—[PAG-X¹]_(n) or multi-functional PAG-X², but notboth, and the covalently stabilized coating can includealginate—[PAG-X¹]_(n), multi-functional PAG-X², or both. In such aninstance, the reacting step can form a surface monolayer by:

reacting alginate—[polyalkalene glycol (PAG)-X¹]_(n) dissolved in theaqueous solution with multi-functional PAG-X² in the covalentlystabilized coating, thereby adding an alginate—[PAG-X¹]_(n) surfacemonolayer to the covalently stabilized coating, or reactingmulti-functional PAG-X² dissolved in the aqueous solution withalginate—[PAG-X¹]_(n), in the covalently stabilized coating, therebyadding a multi-functional PAG-X² surface monolayer to the covalentlystabilized coating.

The forming step can also include rinsing a product of the reacting stepto remove unreacted reactant from the biological material and thenisolating an intermediate biocompatible capsule. Following the rinsingstep, the forming step can include, adding an additional surfacemonolayer to the intermediate biocompatible capsule. If the surfacemonolayer is an alginate—[PAG-X¹]_(n) surface monolayer, the adding stepcan include reacting multi-functional PAG-X² dissolved in an aqueoussolution with the alginate—[PAG-X¹]_(n) in the surface monolayer. If thesurface monolayer is a multi-functional PAG-X² surface monolayer, theadding step can include reacting alginate—[PAG-X¹]_(n) dissolved in anaqueous solution with multi-functional PAG-X² in the surface monolayer.As used herein, “surface monolayer” refers to the molecular monolayer onthe outside of the covalently stabilized coating or the intermediatebiocompatible capsule.

The forming step can also include covalently bonding a base layer of thecovalently stabilized coating to a surface of the biological material.In such an instance, the forming step can include providing an aqueoussolution having the biological material suspended therein, where theaqueous solution includes compound (A) dissolved therein. Compound (A)can then be reacted with amino groups on the surface of the biologicalmaterial. Compound (A) can include:

a first terminal ligand comprising

anda second terminal ligand comprising a ligand selected from the groupconsisting of:

—C≡C;—N₃; and

In the above formulas of (a), (b), (c) and (d), R¹, R² and R³ have thesame meanings used throughout this disclosure.

The forming step, the reacting step, or both can proceed without afree-radical initiator. It has been determined that the step-wiseapplication of polymer layers disclosed herein has at least twobenefits. First, many free-radical initiators, such as those relying onelectromagnetic radiation, can produce moderate to severe cell damage.Second, it is possible to create the encapsulating coating usingmolecular monolayers of alginate and PAG.

It has unexpectedly been discovered that cells encapsulated using thesemolecular monolayers are far superior to previous approaches that relyon bulk polymerization. This is due in part to the superior thicknesscontrol produced using the claimed layer-by-layer formation technique.In addition, although the exact mechanism has yet to be identified, itappears that the molecular monolayer coatings enable implanted cells toavoid rejection while also enabling the implanted cells to receivenutrient, eliminate waste and exchange oxygen. This is evident in FIGS.2 and 3, which show that human and Lewis rat islets, respectively,coated using the techniques disclosed herein survived for more thaneight (8) days after encapsulation with little to no cell deaths. Inaddition, layers formed by this method are superior to an equivalentlayer-by-layer self-assembly via ionic interactions, because suchionically bonded coatings are not stable over the long term.

According to various other embodiments, the coatings can have a highlybranched architectural design. Such coatings can be used to buildprotective coatings on cells, cell clusters, or other bioactive agents,in this way allowing enhanced control over the physical, chemical, andbiological properties of the coating. In addition, the unique branchedsystem provides for a high degree of surface presentation of agents,thereby providing a novel platform for the chemoselective presentationof a large amount of agents on the surface of the coating (which couldbe highly beneficial for targeting, labeling, or as a bioactiveinterface).

Coatings having a highly branched architectural design can be superiorin that: (1) the thickness of the layers can be controlled on anano-scale; (2) the interconnected layers can be covalently linkedresulting in highly stable layers; (3) the multi-branched nature of thefunctionality can result in significant increases in the number offunctional groups available per surface; and (4) the complimentaryelectrostatic charges of the two polymers can enhance the efficiency ofthe procedure and the uniformity of layer deposition.

The highly branched coating systems according to various embodiments canresult in the efficient, convenient, and complete coatings on pancreaticislets of Langerhans with controllable capsule thickness, capsuleproperties, and functionalities helping realize the development ofbioartificial pancreas for the treatment of diabetes. The highlybranched coating systems can be useful for encapsulation of drugs orother loads, as well as the efficient targeting of these agents in vivo,given the high degree of surface functionality of these polymers,multiple tags may be easily presented on the surface of the capsule.Therefore, the highly branched coating systems can provide a high degreeof targeting potential for the capsules that are coated.

Various embodiments rely on the combined use of current, as well as,dendritic and hyperbranched polymeric materials for the assembly offunctional conformal coatings with dendritic-based architectures via thelayer-by-layer film deposition technique.

The nanostructured functional conformal coatings can be deposited ontoviable cells and cell clusters, in addition to other biological relevantsurfaces with nanometer thickness control. The coatings can have athickness within a range having a lower limit and/or an upper limit. Therange can include or exclude the lower limit and/or the upper limit. Thelower limit and/or upper limit can be selected from 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,2000, 3000 nm. For example, according to certain preferred embodiments,the coatings can have a thickness from 0.01 nm to 1000 nm. Thicknessrange is dependent upon desired application.

Chemical functionalities that can be utilized to achieve thelayer-by-layer surface film deposition with added functionality include,but are not limited to, Staudinger ligation, electrostatic interactions,native chemical ligation, Diels-Alder reaction, click chemistries,selective molecular recognition motifs, other non-specificchemical/physical interactions, and combinations thereof.

The functional conformal coatings have many uses, including, but notlimited to, semipermeable physical membranes for the immunoisolation ofcells and functionalization of cell surfaces for particular applicationssuch as molecular recognition, control over biological processes, drugdelivery, and labeling systems for imaging capabilities. Advantages offunctional conformal coatings with dendritic-based architecturesaccording to various embodiments include adequate polymer structuraldesign for maximum control and efficiency over the layer-by-layer filmdeposition, substantial increase in the number of available end groupsfor chemical functionality, and manipulation of structural design ofdeposited films by changing the chemical and physical properties of thedendrimers and hyperbranched polymers.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the examples describedbelow. The following examples are intended to facilitate anunderstanding of the invention and to illustrate the benefits of thepresent invention, but are not intended to limit the scope of theinvention.

EXAMPLES

Customized alginate and PEG (polyethylene glycol) polymers werefabricated to create a chemoselective ligation scheme between thesepolymers based on the Staudinger Ligation chemistry for cellularencapsulation and bioconjugation. As illustrated in the Schemes andFigures below, the azide-alginate [1] is cross-linked via PEG havingeither 2 or 4 DiP end groups ([2] or [3], respectively) to formmacro-gels, micro-gels, or nano-scale layers. PEG linear linkers havingan N-Hydroxysuccinimide (NHS) with an activated carboxylate group at oneend and either a 1-methyl-2diphenylphosphphino-terephthalate (DiP) ([4])or an azide ([5]) group at the other were fabricated for the nano-scalelayering. Bioconjugation capacity was further demonstrated byfluorescently labeling one end of a PEG linear linker and having a DiP([6]) or an azide ([7]) group at the other end. It was found that PEGlinkers bearing a negative charge minimized phase separation andaggregation of the PEG polymer with alginate and may help with anypolymer-related cell toxicity by possibly preventing diffusion of thepolymers across biomembranes. The molecular weights of alginate, PEG, oralginate-PEG polymers were chosen for a particular application in orderto control, viscosity of solution, permeability, cross-linkingpre-incubation time, and nano-layering efficiency. These polymers maythen be used for bioorthogonal encapsulation (via cross-linking) andfunctionalization of cells in macro-scale gels, microcapsules, ornano-scale layers systems.

Materials

Purity, functionalization, and characterization of the polymers wereassessed using attenuated total reflectance Fourier transform infrared(ATR-FT-IR) spectroscopy, mass spectroscopy, and proton nuclear magneticresonance (¹H-NMR) spectroscopy. ATR-FT-IR spectra were obtained usingthe PerkinElmer Spectrum 100 FT-IR spectrometer with the Universal ATR(1 bounce, Di/ZnSe) sampling accessory. UV-Vis spectra were obtainedusing the Molecular Devices SpectraMax M5 Microplate/Cuvette Reader.Samples were submitted to the University of Miami Chemistry Departmentfor mass spectroscopy (VG Trio-2 FAB-MS or Bruker Bioflex IVMALDI-TOF-MS) and ¹H-NMR (Bruker, 400 MHz) analysis. Samples weresubmitted to the North Carolina State University Mass SpectrometryFacility for ESI-TOF-MS analysis. Most chemical reagents were purchasedfrom Sigma-Aldrich at the highest purity available or as indicatedbelow.

Synthesis and Characterization of Alginate-PEG-N₃, Azide-Alg [1]

Ultra purified sodium alginate with high (>60%) guluronic acid contentand high molecular weight (UP-MVG) from NovaMatrix (Pronova Biomedical,Norway) was used as the base polymer for azide functionalization and forcontrol studies of standard alginate capsules. In addition, the very lowviscosity sodium alginate (Pronova UP VLVG, NovaMatrix) wasfunctionalized with azide groups and used as a lower molecular weightalternative for the studies.

H₂N-PEG-N₃ (M_(w) of 372 g/mol by ESI-TOF-MS), the functional groupadded to [1], was fabricated via tosyl-PEG-tosyl and N₃-PEG-N₃ polymerprecursors as shown in Scheme 1, below.

The catalysts and additional reactants used for Scheme 1 are as follows:(i) tosyl(Ts)Cl, 4-(dimethylamino)pyridine (DMAP), dichloromethane(DCM); (ii) NaN₃, N,N-dimethylformamide (DMF); (iiia) Ph₃P, HCl, H₂O,Et₂O, EtOAc; (iii b) Ph₃P, tetrahydrofuran (THF), water. Ts-PEG-Ts wasprepared by tosylation (Ts) of both hydroxyl end groups of HO-PEG-OH(M_(w) of 311 g/mol by ESI-TOF-MS). HO-PEG-OH (2.4 g) was dried bystirring under vacuum at 75° C. for 1 hr. After cooling to roomtemperature (RT, 21-22° C.), the dry PEG oil was dissolved in 3 mLanhydrous dichloromethane (DCM) followed by slow (within 10 min)addition of a freshly prepared solution of 3.94 g (20.46 mmol)p-toluenesulfonyl chloride and 2.80 g (22.69 mmol)4-(dimethylamino)pyridine (DMAP) in 10 mL anhydrous DCM, followed bystirring at RT for 3 h. The resulting mixture was washed once with a 30mL mixture of water/ice/1 mL HCl (36.8%) and once with 20 mL saturatedNaHCO₃ solution containing some ice. The resulting organic layer wasdried over anhydrous Na₂SO₄, filtered through a silica gel pad, and thesolvent removed under reduced pressure. The resulting polymer, Ts-PEG-Ts(3.90 g), was added to NaN₃ (1.27 g), and 4 mL anhydrousN,N-dimethylformamide (DMF) and reacted for 4 h at 80° C. under Ar flowand for 15 h at RT (21-22° C.). Following incubation time, diethyl ether(20 mL) was added. Insoluble salts were removed by filtration through a0.45 μm polypropylene filter, diethyl ether was removed under reducedpressure, and the DMF was removed by short-path distillation at 80° C.with help of Ar flow. The residue was dissolved in 20 mL diethyl etherand passed through a silica gel pad. The diethyl ether was removed underreduced pressure. The product, N₃-PEG-N₃ was dried first by Ar-flushingand then under reduced pressure. A solution of triphenylphosphine (Ph₃P)(1.06 g) in 9 mL of 50% (v/v) diethyl ether in ethyl acetate was slowly(within 1 h) added to a solution of N₃-PEG-N₃ (1.5 g), 50% (v/v) diethylether (4.5 mL) in ethyl acetate, and 0.5M HCl_(aq) (9 mL) under Ar-flowand rapid stirring. Solution was then stirred for 19 h at RT. Theaqueous phase was collected and made basic with addition of 6M NaOH_(aq)(1.0 mL). The solution was then cooled in ice-water bath for 2 h whilebubbling Ar, passed through a 0.45 μm polypropylene filter, andextracted 7 times with 3 mL dichloromethane (DCM). All combined DCM wasdried over anhydrous Na₂SO₄, passed through a silica gel pad, and DCMremoved under reduced pressure followed by Ar-flushing. Method asoutlined resulted in 1.0 g of H₂N-PEG-N₃

Scheme 2, below, shows the fabrication of alginate-PEG-N₃ [1].

In scheme 2, the catalysts and additional reactants are as follows: (i)EDC, NHS, NH₂—PEG-N₃. In Scheme 2, sodium alginate (50 mg) (UP-MVG),N-hydroxysuccinimide (14 mg, 0.12 mmol), and2-(N-morpholino)ethanesulfonic acid (62 mg, 0.29 mmol) were dissolved in5 mL deionized water. A solution of H₂N-PEG-N₃ (28 mg, 0.08 mmol) in 200μL H₂O was added.

After stirring for 10 min at RT,1-ethyl-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) was addedand stirring continued for 25 min at RT. The amount of EDC added variedfrom 56 mg to 167 mg, depending on the degree of functionalization (from3 to 13% by H¹-NMR and ATR-FT-IR) desired. For this study, it was foundthat the addition of 116 mg of EDC resulted in high (9-12%) degree ofH₂N-PEG-N₃ functionalization. This alginate was used for allencapsulation experiments in order to minimize possible EDC-related sidereactions with alginate. Following incubation, 55 μL of 6 M NaOH wasadded and stirred for an additional 10 min. Purification was achieved by(at least) 4 days dialysis (10,000 MWCO membrane) against 600 mL waterchanged three times a day. During the first 3 days, 10 μL of 6M NaOH and400 μL 4.26M NaCl was added twice daily to the alginate-containingsolution and mixed. Finally, solution was filtered through a 0.45 μmmembrane and lyophilized. The yield was 49 mg to 56 mg of a white,foamy, and solid material. The Kaiser test was negative. ATR-FT-IR:3279, 2878, 2113, 1658 (shoulder), 1601, 1547 (shoulder) cm⁻¹. ¹H-NMR(D₂O): δ 3.45-3.93 (s-m, 1H, PEG) and 4.67-4.95 (m; 5H or 2H foralginate with 3-5 or 10-13% modification, respectively).

The same above procedure was followed to synthesis Azide-Alginate havinglower molecular weight alginate except that 50 mg of UP-VLVG alginatewas used instead. For the fluorescent labeling of [1] (Azide-Alg-CF[1]), 20 mg of H₂N-PEG-CF (synthesized as part of compound [6] and [7])was also mixed with the alginate prior to addition of EDC.

Synthesis of Poly(ethylene glycol) (PEG) Diphosphine, 2-DiP-PEG [2]

Scheme 3, below, shows a pathway for synthesis of Poly(ethylene glycol)(PEG) diphosphine, called 2-DiP-PEG.

In Scheme 3, the catalysts and additional reactants are as follows: (i)HCl, NaNO₂; (ii) KI; (iii) Pd(Ac)₂, Ph₂PH, acetonitrile. Poly(ethyleneglycol) (PEG) diphosphine, called 2-DiP-PEG [2] was fabricated from thestarting PEG polymer of NH₂—PEG-NH₂ (M_(w) of 3513 g/mol byMALDI-TOF-MS). Functionalization of the end units of the PEG wasachieved by first fabricating the phosphine group or1-methyl-2-diphenylphosphino-terephthalate (DiP), Scheme 3. DiP wasfabricated by the addition of 1-Methyl-2-aminoterephthalate (1.0 g, 5.10mmol) to 10 mL of cooled HCl and stirred 10 min. NaNO₂ (0.36 g, 5.22mmol) in 2.3 mL water was injected slowly (within 5 min) through asealed rubber septum into the HCl mixture while stirring (an orange gasformed). After 5 min, the mixture was removed from the ice-water bathand stirred 25 min at RT. The resulting mixture was passed through glasswool and 0.45 μm polypropylene (pp) membrane filter into a stirringsolution of (8.6 g, 51.7 mmol) Kl and 14 mL water (mixture turns darkred with red-black precipitate). After stirring for 1 h at RT, thesolution was mixed with 40 mL DCM and 35 mL of 1.57 M (55.0 mmol)Na₂SO₃. The DCM phase was collected and washed once with 35 mL of 4.28MNaCl solution. The DCM phase was collect, dried over anhydrous Na₂SO₄,filtered (0.45 μm, pp membrane), and DCM removed under reduced pressure.The yellow solid residue was dissolved in 6 mL methanol and filtered(0.45 μm, pp membrane). The product was precipitated by adding 4 mLwater, cooling 1 h in ice-water bath, and stirring. It was collected byfiltration and freeze-dried overnight to yield 832.0 mg of a yellowsolid powder of 1-methyl-2-iodoterephthalate. A flame dried/Ar-flushedSchlenk tube was loaded with 1-methyl-2-iodoterephthalate (750 mg,2.4506 mmol), Pd(Ac)₂ (5.8 mg, 0.0256 mmol), anhydrous acetonitrile (7.5mL), and N,N′-diisopropylethylamine (900 μL, 5.1465 mmol). The mixturewas stirred until fully dissolved and degassed (freeze-pump-thawmethod). Diphenylphosphine (430 μL, 2.4489 mmol) was injected(drop-wise) into the acetonitrile solution under Ar flow (solutionturned red and clear). The solution was refluxed for 4 h under Arpressure (balloon), cooled to RT (or incubated overnight at RT), andconcentrated under reduced pressure. The red solid-oily stick massresidue was dissolved in 50 mL DCM and washed once with 25 mL water and25 mL 1M HCl. The DCM was removed under reduced pressure. The productwas recovered by washing the red-yellow solid residue with 10 mL coldmethanol, rinsed 5×2 mL cold methanol, and filtration. After dryingunder reduced pressure, 656.8 mg of a yellow solid powder of DiP (Mw of364.09 g/mol by ESI-TOF-MS) was obtained. 2-DiP-PEG [2] as shown inScheme 4 was formed by the addition of N,N′-diisopropylcarbodiimide(DIC) (22.524, 0.1440 mmol, DIC) to a solution of DiP (52.8 mg, 0.1449mmol) and N-hydroxysuccinimide (17.2 mg, 0.1465 mmol) in 1.2 mL DMF(anhydrous, degassed with Ar). After stirring for 30 min at RT under Arflow, NH₂—PEG-NH₂ (200 mg, 0.0597 mmol) was added, followed by4-dimethylaminopyridine (14.8 mg, 0.1200 mmol) after 1 hr. After 3 hrs,stirring at RT under Ar flow, the product was precipitated with 12 mLcold diethyl ether and collected by centrifugation at 0° C. It was“crystallized” once in 10 mL 200 proof ethanol (warm to 37° C. todissolve, cool in ice-water bath to precipitate, collect precipitate bycentrifugation at 0° C.). It was dissolved/precipitated/collected oncewith 0.5 mL DCM/12 mL cold diethyl ether/centrifugation at 0° C. anddried under reduced pressure. The yield was 212.0 mg of a white to lightyellow solid powder. The Kaiser test was negative. M_(w) of [2]: 4057g/mol by MALDI-TOF-MS. ¹H-NMR (CDCl₃): δ 3.45-3.84 (m, 393H), 6.80 (s,2H), 7.28-7.38 (m, 22H), 7.80-7.82 (dt, 2H, J=2.0, 1.8, 8.2 Hz), and8.06-8.09 (m, 2H).

Synthesis of 4-DiP-PEG [3]

Scheme 4 shows the synthesis of multi-functional PEG molecules forcross-linking of alginate via the Staudinger ligation reaction.

In Scheme 4, the catalysts and additional reactants are as follows: (i)N,N′-diisopropylcarbodiimide (DIC), NHS; (ii) NH₂—PEG-NH₂, DMAP. Allamino end groups of a 4arm-PEG (M_(w) of 11,000 g/mol) werefunctionalized with aspartic acid (Asp) followed by DiP resulting in anegatively charged polymer bearing four reactive sites for efficientcross-linking or layering, Scheme 4. 4DiP-PEG [3] was synthesized byfirst reacting H₂N-4arm-PEG (300 mg, 0.027 mmol), Fmoc-Asp(OtBu)-NHS (64mg, 0.126 mmol), and N,N′-diisopropylethylamine (24 μL, 0.137 mmol) in1.2 mL anhydrous DMF for 30 min at RT. Purification was achieved byprecipitating with 10 mL cold (ice-water bath) Et₂O, “crystallizing” in10 mL EtOH (37° C. to dissolve then cool in ice-water bath toprecipitate), and washing with 10 mL cold Et₂O. All precipitates werecollected by centrifugation (3500 rpm, 0° C., 5 min). The precipitatewas dried under vacuum to yield 311.7 mg of [Fmoc-Asp(OtBu)]₄-PEG as ayellow-green to white solid powder. Kaiser test: negative. ATR-FT-IR:3534, 3311, 2883, 1727, 1675, 1533, 1099, 762, 742 cm⁻¹. Second,[Fmoc-Asp(OtBu)]₄-PEG (270 mg, 0.022 mmol) was dissolved in 1.2 mL of20% piperidine in DMF and reacted 30 min at RT. The product,[H₂N-Asp(OtBu)]₄-PEG, was precipitated with 10 mL cold (ice-water bath)Et₂O and collected by centrifugation (3500 rpm, 0° C., 5 min). It wasthen dissolved in 6 mL EtOH at 37° C. followed by addition of 2 mL Et₂O,cooling in ice-water bath to re-precipitate the product and collected bycentrifugation as above. It was washed with 10 mL cold Et₂O and driedunder vacuum to yield 217.4 mg of a green to white solid powder. Kaisertest: positive. ATR-FT-IR: 3523, 3384, 2885, 1725, 1670, 1520, 1098cm⁻¹. Third, [H₂N-Asp(OtBu)]₄-PEG (165 mg, 0.014 mmol), DiP-NHS (34 mg,0.093 mmol), DMAP (7 mg), triethylamine (32 μL, 0.23 mmol), and 700 μLanhydrous DMF were reacted 23 h at RT. The product,[DiP-Asp(OtBu)]₄-PEG, was purified as in the second step above. To yield165.7 mg of a green to white solid powder. Kaiser test: negative.ATR-FT-IR: 3508, 3324, 2883, 1721, 1665, 1533, 1101, 748, 699 cm⁻¹.Finally, [DiP-Asp(OtBu)]₄-PEG (145 mg, 0.011 mmol) was reacted in a DCMsolution containing 5% (v/v) triisopropylsilane and 35% (v/v)trifluoroacetic acid for 3 h at RT. The polymer was precipitated andwashed once with 10 mL cold Et₂O followed by drying under vacuum. Thedried product was dissolved in 400 μL DMF containing 37 μL oftriethylamine and stirred well. Cold Et₂O (10 mL) was added toprecipitate the polymer, which was then dissolved in 6 mL EtOH at 37° C.and cooled in ice-water bath to re-precipitate. The precipitate waswashed once with 10 mL cold Et₂O and dried under vacuum. The resultingpolymer was dissolved in 1 mL water and passed through a 4 mL column ofSP-Sephadex C-25 using water as solvent and freeze-dried to yield 123.3mg of [DiP-Asp(Na⁺)]₄-PEG or [3], a green-white solid powder. ATR-FT-IR:3511, 2883, 1720, 1658, 1589, 1537, 1095, 749, 699 cm⁻¹.

This polymer was also prepared as above but without Asp (aspartic acid)for comparison.

Synthesis of DiP-PEG-NHS [4]

The structure of DiP-PEG-NHS [4] is shown below.

It was prepared in two steps process. First, the DiP-NHS (26.8 mg) wasreacted with H₂N-PEG-COOH (200 mg, from Laysan Bio, M_(w) 3400 g/mol)and triethylamine (31.8 μL) in 800 μL anhydrous DMF for 3 h at roomtemperature (RT) under Ar. The resulting polymer, DiP-PEG-COOH, wascollected by precipitation with 10 mL cold (ice-water bath) Et₂O andcollected by centrifugation (3500 rpm, 5 min, 0° C.). It was then“crystallized” three times in 8 mL EtOH (dissolved at 37° C. andprecipitated by cooling in ice-water bath), washed with 10 mL Et₂O, anddried under vacuum. Yield: 187 mg of a white, solid powder. Kaiser test:negative. ATR-FT-IR: 2884, 1656, 1101, 1538, 749, 699 cm⁻¹. Second, 180mg of DiP-PEG-COOH was reacted with 15.8 mg NHS and 21.1 μLdiisopropylcarbodiimide in 700 μL anhydrous DMF for 3 h under Ar. Theproduct was collected and purified as in the first step except thatcrystallization was performed only once. This activation reaction wasrepeated once more. Yield: 155 mg of a white, solid powder. ATR-FT-IR:2884, 1812, 1783, 1738, 1660, 1102, 1543, 750, 700 cm⁻¹.

Synthesis of Azide-PEG-NHS 151

The structure of Azide-PEG-NHS [5] is shown below.

The synthesis of Azide-PEG-NHS consisted of five steps: (1) H₂N-PEG-COOH(300 mg, Laysan Bio, M_(w) 3400 g/mol) was reacted with 50 mgFmoc-Asp(OtBu)-NHS and 19 μL N,N′-diisopropylethylamine in 1 mLanhydrous DMF for 30 min at RT under Ar. Cold Et₂O (10 mL) was addedwith vortex to precipitate the product, which was collected bycentrifugation. It was dissolved in 6 mL EtOH at 37° C. andre-precipitate by cooling in ice-water bath. After washing with 10 mLEt₂O, it was dried under vacuum. Yield: 315 mg of a white, solid powder.Kaiser test: negative. ATR-FT-IR: 3516, 3317, 2884, 1727, 1668, 1539,1101, 762, 743 cm⁻¹. (2) Product from step 1 (290 mg) was reacted with1.2 mL of 20% piperidine in DMF for 30 min. Purification was achieved asin step 1 with the addition that the dried powder was dissolved in 1 mLwater, passed through a 4 mL SP-Sephadex C-25 column using water andfreeze-dried. Yield: 270 mg of a white, solid powder. Kaiser test:positive. ATR-FT-IR: 3505, 3330, 2884, 1725, 1659, 1550, 1100 cm⁻¹. (3)Product from step 2 (250 mg) was reacted with 70 mg of azide-PEG-NHS(M_(w) 835 g/mol), 3 mg N,N′-dimethylaminopyridine, and 39 μLtriethylamine in 700 μl DMF for 23 h at RT under Ar. Product wascollected and purified as in step 1. Yield: 248 mg of a white, solidpowder. Kaiser test: negative. ATR-FT-IR 3519, 3311, 2884, 2105, 1729,1654, 1545, 1100 cm⁻¹. (4) Product from step 3 (230 mg) was reacted with18 mg NHS and 24 μL of diisopropylcarbodiimide in 600 μL DMF for 2 h atRT under Ar. This activation step was repeated once more. The productwas collected and purified as in step 1. Yield: 208 mg of a white, solidpowder. ATR-FT-IR: 3516, 3320, 2885, 2107, 1812, 1782, 1737, 1658, 1545,1100 cm⁻¹. (5) Product from step 4 (190 mg) was reacted with 1 mL of DCMsolution containing 5% v/v triisopropylsilane and 35° A) v/vtrifluoroacetic acid for 3 h at RT under Ar. Product was collected andpurified as in step 1. Yield: 172 mg of a white solid powder. ATR-FT-IR:3524, 3319, 2885, 2105, 1812, 1783, 1737, 1659, 1543, 1100 cm⁻¹.

Synthesis of DiP-PEG-CF [6]

The structure of DiP-PEG-CF [6] is shown below.

The synthesis of DiP-PEG-carboxyfluorescein(CF) consisted of two mainsteps: (1) H₂N-PEG-NH₂ (200 mg, M_(w) 3513 g/mol) was reacted with5(6)-carboxyfluorescein-NHS (28 mg, M_(w) 473.39) and triethylamine(15.9 μL) in 1 mL anhydrous DMF for 20 min at RT under Ar in the dark.Cold (ice-water bath) Et₂O (10 mL) was added to precipitate the polymerand was collected by centrifugation (3500 rpm, 5 min, 0° C.). Theprecipitate was dissolved in 8 mL EtOH with vortex at 37° C. followed bycooling in ice-water bath to re-precipitate and centrifuged as above tocollect the precipitate. After washing the precipitate with 10 mL coldEt₂O, it was dried under reduced pressure. The dry polymer was dissolvedin 1.5 mL water and passed through an 8 mL column of SP-Sephadex C-25followed by freeze-drying. This last purification step was repeated butusing a QAE Sephadex column instead. Finally, it was dissolved in 3 mLDCM filtered through a 0.45 μm membrane filter, precipitated with 20 mLcold Et₂O, and dried under reduced pressure. Yield: 107 mg of ayellow-orange, solid powder. Kaiser test: positive. ATR-FT-IR: 2884,1658, 1615, 1546, 1100 cm⁻¹. (2) Product from step 1 (70 mg) was reactedwith 9 mg of DiP-NHS and 10.4 μL triethylamine in 280 μL anhydrous DMFfor 2 h at RT under Ar. Cold Et₂O (5 mL) was added to precipitate theproduct. It was dissolved in 5 mL EtOH at 37° C., cooled in ice-waterbath to precipitate, collected by centrifugation as above, washed withEt₂O, and dried under reduced pressure. The dry polymer was dissolved in1 mL water, passed through a 3 mL SP-Sephadex C-25 column using water assolvent, freeze-dried, dissolved in 5 mL EtOH at 37° C., filteredthrough a 0.45 μm membrane filter, cooled in ice-water bath toprecipitate, washed with 5 mL cold Et₂O, and dried under reducedpressure. Yield: 63 mg of an orange solid powder. Kaiser test: negative.ATR-FT-IR: 2885, 1758, 1721, 1656, 1613, 1548, 1099, 750, 699 cm⁻¹.M_(w): 3982 g/mol by MALDI-MS.

Synthesis of Azide-PEG-CF [7]

The structure of Azide-PEG-CF [7] is shown below.

The synthesis of Azied-PEG-CF consisted of two main steps: (1) The samestep 1 as described for compound [6]. (2) The product from step 1 (90mg) was reacted with 19.2 μL N₃-PEG-NHS (M_(w) 835 g/mol) and 12.8 μLtriethylamine in 350 μL anhydrous DMF for 30 min at RT under Ar in thedark. The product was purified as described in step 2 for compound [6].Yield: 93 mg of an orange, solid powder. Kaiser test: negative.ATR-FT-IR: 2883, 2099, 1656, 1611, 1572, 1546, 1097 cm⁻¹.

Microcapsule Formation and Cellular Encapsulation

Cross-linked Alginate-PEG (XAlg-PEG) microbeads were fabricated using amodification of the protocol originally developed by Sun (1). XAlg-PEGgels consisted of 2.5 wt % Azide-Alg [1] and 4.75 wt % 2-DiP-PEG [2].The solution of [1] and [2] were generated by first dissolving 3.1 wt %of M in saline in one vial and 23.75 wt % of [2] in another. Followingcomplete dissolution of the polymers in saline, the solutions were mixedin a 1:1.9 ratio. The mixture of [1] and [2] were gently mixed for 1 hr15 mins prior to microbead fabrication. Microbeads were prepared vianeedle extrusion of the XAlg-PEG solution into a gelling basin of 50 mMBaCl₂ (Sigma Aldrich) and 0.025% (v/v) Tween 20 (Sigma Aldrich)solution. The size of the droplets was controlled by a parallel airflowgenerator (10 kPa pressure of air with a 1 in distance from the needleto the gelling solution) and the manual force applied to the syringe.The beads were exposed to the gelation solution for 5 min, aspirated,and then rinsed with phosphate-buffered solution (PBS) three times toremove excess barium. For microcapsules containing cells, cells weremixed in either polymer solution immediately prior to needle extrusion.Following the homogeneous distribution of the cells within either theXAlg-PEG solutions, the cell/polymer suspension was extruded through themicroencapsulation generator and microbeads were fabricated. Following a5 min exposure to the barium gelation solution and PBS rinse, thecell-containing microbeads were washed in the appropriate culture mediafor the cell type.

FIGS. 1-4 show microcapsules used for encapsulating cell lines andislets as disclosed herein. In FIG. 1, light lines represent Azide-Algmolecules [1] and the darker lines represent the 2-Dip-PEG molecules[2]. Covalent linkages can be established in pre-incubation time,followed by the exposure to barium ions during capsules formation toform a gel instantaneously. After ionic gel formation, covalentcross-linking between the two reactive polymers continues to “lock” thegel in place.

FIGS. 2( a)-(c) show confocal images of Lewis rat islets encapsulated asdisclosed herein stained such that live cells are green whereas deadcells are red. FIGS. 2(A), (B) and (C) show the Lewis rat islets onpost-encapsulation Day 1, Day 5, and Day 8, respectively.

FIGS. 2( a)-(c) show confocal images of human islets encapsulated asdisclosed herein stained such that live cells are green whereas deadcells are red. FIGS. 2(A), (B) and (C) show the human islets onpost-encapsulation Day 1, Day 5, and Day 8, respectively.

In addition, ionic (Ba²⁺), ionic/covalent, and covalent crosslinkedbeads were also prepared with [1] and [3] utilizing only 1.5 wt % ofboth polymers (FIG. 5). For the covalent cross-linking, a pre-incubationperiod of only 45 min was utilized. An advantage of compound [3] is moresoluble polymeric mixtures. It is envisioned that the pre-incubationtime can be eliminated (if needed) by designing higher molecular weightpolymers (PEG or alginate/PEG) bearing the phosphine groups for theStaudinger ligation.

Nano-Scale Layer Fabrication and Cellular Encapsulation

Nano-scale layers were fabricated on glass microcarrier beads of sizeranging from 150 to 210 μm. The beads were cleaned with piranha solution(mixture of 3:1 concentrate sulfuric acid and hydrogen peroxide) for 20minutes and then washed extensively with DI water. The glass beads werethen amino functionalized by a 5% wt solution of4-aminobutyl-triethoxy-silane in toluene at 80° C. for 5 hours to mimicthe amino groups on the cell surface. Amino functionalized glass beadswere incubated in a 0.5 to 5 mM solution of Azide-PEG-NHS [5] for 1 hourto initiate the first layer of poly(ethylene glycol). Following the PEGcoating, the beads were washed to remove the non-specific adsorbedpolymer and incubated again in a 0.7 mM solution of 4-DiP-PEG [3] for 45minutes to form a second layer of multi-arm PEG. After washing thebeads, they were incubated again in a 0.2 to 2 mM of Azide-Alg [1] orfluorescent labeled Azide-Alg-CF [1a] for 1 to 2 hours to form a thirdlayer of polymer. Additional layers are added by alternating the4-DiP-PEG [3] and Azide-Alg [1] or Azide-Alg-CF [1a]. A nanocapsule wasalso made by incubating the amino functionalized glass beads withAzide-PEG-NHS [5] and then in a mixture of 4-DiP-PEG [3] and Azide-Alg[1] or Azide-Alg-CF [1a] (0.7 and 0.2 mM respectively for 2 hrs) to forma multilayer capsule.

FIG. 6(A) shows fluorescent microscope image of amino functionalizedglass microcarrier beads following incubation with NHS-PEG-CH₃ (LaysanBio, M_(w) 5,000 g/mol) (2.5 mM, 1 hr), rinse with Dulbecco modifiedphosphate buffer saline (DPBS) 1× buffer, subsequent incubation with4-DiP-PEG [3] (0.7 mM, 45 minutes), another rinse, and final incubationof Azide-Alg-CF [1a] (0.2 mM and 2 hrs) and a rinse with buffer wereperformed. The lack of fluorescent on the glass beads surface is due tothe absence 4-Dip-PEG and azide-Alginate-CF layer (Control experiment).

FIG. 6(B) shows fluorescent images of amino functionalized glassmicrocarrier beads following incubation with Azide-PEG-NHS [5] (2.5 mM,1 hr), rinse, subsequent incubation with 4-DiP-PEG [3] (0.7 mM, 45minutes), another rinse, and final incubation of Azide-Alg-CF [1a] (0.2mM and 2 hrs) and final rinse with buffer. The third layer of polymerwas labeled with FITC and all the layers are all covalent bond andstable even after five weeks in buffer solution.

FIG. 6(C) shows fluorescent images of amino functionalized glassmicrocarrier beads following incubation with Azide-PEG-NHS [5] (2.5 mM,1 hr), rinse, subsequent incubation with a mixture of 4-DiP-PEG [3] andAzide-Alg-CF [1a] (0.7 mM and 0.2 mM, 2 hrs) and a final rinse withbuffer.

FIGS. 7(A)-(F) show confocal fluorescent and light transmission imagesof nano-layering on glass microcarrier beads (170 to 210 μm glass beadswere functionalized with 4-aminobutyl-triethoxy-silane) reacted withfunctionalized polymers.

FIGS. 7(A) and (B) show confocal images, fluorescent and lighttransmission, respectively, of amino functionalized glass microcarrierbeads following incubation with NHS-PEG-CH₃ (2.5 mM, 1 hr), rinsed withbuffer and subsequent incubation with 4-DiP-PEG [3] (0.7 mM, 45 minutes)and wash and final incubation of Azide-Alg-CF [1a](0.2 mM and 2 hrs) anda final rinse with buffer. The lack of Azide-Alg-CF bounded to the glasssurface is due to the absence of the 4-DiP-PEG and Azide-Alg-CF polymer(Control experiment)

FIGS. 7(C) and (D) show confocal images, fluorescent and lighttransmission, respectively, of amino functionalized glass microcarrierbeads following incubation with Azide-PEG-NHS [5] (2.5 mM, 1 hr),followed with intensively rinsing with buffer. A Subsequent incubationwith 4-DiP-PEG [3] (0.7 mM, 45 minutes), rinsing and final incubation ofAzide-Alg-CF [1a] (0.2 mM and 2 hrs), and final rinsed with buffer wasformed. The third layer of polymer was labeled with FITC which is stableand covalent bounded to the surface after five weeks in buffer.

FIGS. 7(E) and (F) show confocal images, fluorescent and lighttransmission, respectively, of amino functionalized glass microcarrierbeads following incubation with Azide-PEG-NHS [5](2.5 mM, 1 hr),followed by rinsing with buffer and a subsequent incubation with amixture of 4-DiP-PEG [3] and Azide-Alg-CF [1a] (0.7 mM, 0.2 mM, 2 hrs)and a final rinse with buffer. Multiple layers are formed after mixingthe functionalized beads with 4-DiP-PEG [3] and Azide-Alg-CF [1a],resulting in a much thicker coating.

The morphology of each layer was studied by Atomic Force Microscopy(AFM). The nano-scale layers were prepared on a Corning amino glassslide following the same procedure for the nano-scale layer fabrication.Figures (A) and (B) are height and deflection images, respectively, ofCorning amino glass functionalized with a layer of Azide-PEG-NHS [5];while Figures (C) and (D) are height and deflection images,respectively, of Corning amino glass functionalized with a layer ofAzide-PEG-NHS [5] followed by a layer of 4-DiP-PEG [3]. As shown inFIGS. 8(A)-(D), the surface morphology was found to be smooth, with noholes or de-wetting observed.

Cells are incubated in a 4 mM solution of NHS-PEG-DiP for 30 mins toinitiate first layer of poly(ethylene)glycol. Following PEG coating,cells are then incubated with 0.5% Azide-Alg [1] for 1 hr and washed.After alginate coating, cells are then incubated with 2-DiP-PEG [2] for1 hr. Additional layers may be added by alternating layers of Azide-Alg[1] and 2-DiP-PEG [2] (or 4-DiP-PEG [3]) with washes in between eachpolymer coating step.

FIGS. 9 and 10 show schematics of monolayer coating schemes. FIG. 9shows a schematic representation of covalently linked layers oftriarylphosphine PEG active ester(base layer),azido-alginate(interconnecting layer) and bi-triarylphosphine PEG(interconnecting layer and terminal layer).

FIG. 10 shows a hemical scheme outlining the reactions for the covalentlinking of two layers (triarylphosphine PEG active ester andazido-alginate) on the islet surface. Alternatively, the azide group canbe first linked to islet surface using compound [5] followed byalternate layers of compounds [3] and [1], image not shown.

FIGS. 11(A)-(D) show confocal images of Cytodex-3 beads (dextran beadscoated with a ˜20-30 μm layer of denatured collagen) treated withfunctionalized polymers. FIGS. 11(A) and (C) are fluorescent and lighttransmission images, respectively, of collagen beads followingincubation with DiP-PEG-NHS [4] (2.5 mM, 30 mins) and azide-PEG-CF [7]label (2.5 mM, 2 hrs). FIGS. 11(B) and (D) are fluorescent and lighttransmission images, respectively, of collagen beads followingincubation with PEG-NHS (2.5 mM, 30 mins) and azide-PEG-CF label (2.5mM, 2 hrs). Images illustrate selectivity of azide-PEG-CF reaction totriarylphosphine PEG and intensity of binding. The thick scale of thecoating is due to the thickness of the collagen layer on the dextranbeads, which is approximately 20-30 μm thick, where the active ester ofthe PEG polymers conjugates with the free amines throughout the layer.

FIGS. 12(A) and (B) show confocal images of human islets treated withthe functionalized polymers disclosed herein. Fluorescent confocalimages of human islets following incubation with triarylphosphine PEGactive ester [4] (2.5 mM, 30 mins) and azide-PEG-CF [7] label (2.5 mM, 2hrs), where FIG. 12(A) is the projection of a z-stack image for the tophalf of the islet and FIG. 12(B) is an image of a single slice throughthe mid-section of the islet. Islets incubated with PEG active ester(2.5 mM, 30 mins) and azide-PEG-CF labels (2.5 mM, 2 hrs) did notexhibit fluorescence. Presumably, this is because the absence of thetriarylphosphine on the PEG polymer prevented CF binding.

Macro-Scale Gel Formation.

XAlg-PEG gels consisted of 2.50 wt % Azide-Alg [1] and 2.5-4.75 wt %2-DiP-PEG [2] (or 4-DiP-PEG [3]). The solution of [1] and [2] weregenerated by first dissolving 3.1 wt % of [1] in saline in one vial and23.75 wt % of [2] in another. Following complete dissolution of thepolymers in saline, the solutions were mixed in a 1:1.0-1.9 ratio andmixed with cells. Gels were poured into molds or extruded through tubingand incubated for 5 hrs or until complete gel formation. The resultinggels could not be dissolved in EDTA and showed minimal swelling invarious ionic solutions. FIG. 13 shows images of macro-scale gelsproduced as described above in the form of capsules, cylinders anddisks.

Formation of a Highly Branched Coating System

A dendritic-based coating was accomplished by a combination ofpoly(amido amine) (PAMAM) dendrimer and novel hyperbranched alginatefunctionalized with complementary end groups for Staudinger ligation,which is a highly chemoselective and hence desirable covalent ligationscheme for cellular work. To prepare these materials, PAMAM ofgeneration 5 was utilized to introduce one of the layers. Based on itsdendritic structure, this PAMAM has 128 primary amino end groupsavailable for reaction that were readily reacted with 1-methyl4-pentafluorophenyl diester (MDT) in dichloromethane for 15-60 min.After purification by dissolution and precipitation using 50% methanolin dichloromethane and diethyl ether, the product was purified bydialysis and freeze dried. Glutaric anhydride was used to manipulate thenet surface charge of PAMAM dendrimer in the presence of triethylaminebase. PAMAM dendrimers having 15-40% of the end groups modified with MDTand surface net charge ranging from +110 to −38 were prepared andcharacterized by infrared and proton nuclear magnetic resonance (NMR)spectroscopies. For the other alternate complimentary polymer layer,hyperbranched alginate functionalized with azido (N3) end groups wasprepared. Carbodiimide condensation of 3,5-dicarboxyphenyl glycineamidein the presence of alginate was utilized to grow the hyperbranches onthe alginate polymeric backbone, followed by dissolution andprecipitation in aqueous solution and acetone for purification anddrying under reduced pressure. Due to free carboxylate end groups, thehyperbranched alginate is negatively charged. These carboxylate groupswere utilized to functionalize the polymer with azido end groups viapoly(ethylene glycol) linkers and characterized by infrared and protonNMR spectroscopies.

Graphical illustrations of these two highly branched and functionalpolymers are shown in FIGS. 14A and 14B. More specifically, FIG. 14Ashows a functionalized PAMAM dendrimer, where generation 2 is shown forsimplicity. FIG. 14B shows an azido-functionalized hyperbranchedalginate.

Alternate deposition of these two polymeric materials (2 mg/mL, variousaqueous buffers) resulted in the successful encapsulation of ratpancreatic islets as shown in the fluorescence image on FIG. 15. Morespecifically, FIG. 15 shows a confocal cross-section image revealing thefluorescent capsule of 6 layers deposited around on rat pancreaticislets. These capsules can serve as semipermeable protective barriersaiming to aid immunoisolation of cells during islet transplantationprocedures without imposing significant thicknesses to impede nutrientdiffusion. Fluorescent labeling of the polymers permit evaluation of thecapacity of these polymers for variety of chemical functionality in onesystem arising from large number of available functional groups on thebranched materials. Either covalent of a combination of covalent andelectrostatic interactions were utilized to assemble the coatings.Multilayer coatings were stable over time. Minor cytotoxicity wasobserved when using the positively charged polymers, which is common forpolycations; however, cell viability was improved by decreasing polymerconcentration, net positive charge, and incubation time.

Coated islets remained functional, as they released insulin in responseto glucose (FIG. 15) and were functionally comparable to naked isletseven after 4 h of stimulated insulin release.

FIG. 16 shows a amount of insulin released by islets upon incubation inlow (60 mg/dL), high (300 mg/dL), and low glucose containing buffer for1 h each. Uncoated islets (group 1) and islets coated with 6 layers(group 2) are shown.

FIG. 17 shows an electron microscopic image of a 12 layer film depositedon Si wafer.

To further evaluate such coating films, multiple layers were depositedon Si wafer resulting in highly homogeneous film topographies (FIG. 16)and linear (R2 0.99) increase on film thickness with each depositedlayer (FIG. 17). Si wafers were azido functionalized via silanizationwith 11-bromoundecyltrichlorosilane followed by sodium azide treatment.Washing of wafers with highly ionic solutions did not affect the polymerthickness, illustrating the superior stability of these convalentlylinked layers.

FIG. 18 shows the film thickness increase upon deposition of a bilayeron the azido-functionalized Si wafer. A bilayer consisted of one layerof the functionalized PAMAM dendrimer and one layer of thefunctionalized hyperbranched alginate.

It is to be understood that while the invention in has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples which follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

We claim:
 1. A protective coating for covering a biological material, the protective coating comprising a plurality of interconnected layers covalently bonded to each other, wherein the plurality of interconnected layers comprise: at least one hyperbranched alginate, and at least one dendrimer.
 2. The protective coating of claim 1, further comprising at least one biologically-active agent chemoselectively presented on a surface of the coating.
 3. The protective coating of claim 2, wherein the biologically-active agent is one selected from a targeting agent, a labeling agent, a bioactive interface, and combinations thereof.
 4. The protective coating of claim 1, wherein the biological material is one selected from the group consisting of a plurality of cells, a plurality of cell clusters, a plurality of bioactive agents, and combinations thereof.
 5. The protective coating of claim 1, wherein the at least one hyperbranched alginate comprises an azido-functionalized hyperbranched alginate.
 6. The protective coating of claim 1, wherein the at least one dendrimer comprises a poly(amido)amine dendrimer.
 7. The protective coating of claim 5, wherein the at least one dendrimer comprises a poly(amido)amine dendrimer. 